Method and apparatus for disruption of biological material

ABSTRACT

A method of disrupting biological material includes drying particulate material, mixing the material with a gas under pressure, releasing the pressure explosively and collecting the resultant product. The biological starting material is any particulate material and includes: cells with membranes, cells with rigid cell walls, non-cellular biological material, intra-cellular material, and unbounded homogenous material. Apparatus for batch, semi-continuous and continuous operation of the method is provided. Included is a chamber with at least one inlet valve and at least one outlet valve and collection means. The chamber is capable of withstanding at least 800 bar, preferably 30 bar pressure. The particle size of the starting material is in the range 0.1 to 2000 μm and of the resultant product, less than 2 μm.

TECHNICAL FIELD

The present invention relates to apparatus for the explosivedecompression of biological material, and to a method of explosivedecompression of biological material. More specifically, the apparatusand method relate to the explosive decompression of biological materialresulting in a product that is homogenous in size.

BACKGROUND ART

Various methods of disrupting cell walls of biological material areknown, the method (and associated apparatus) depending on whether thecell walls are rigid, elastic, neither, or do not exist. For example,sonic disruption of cells is used on non-rigid materials. However thisis not effective on cells with rigid cell walls.

Mechanical methods for disrupting cells of biological material, such asgrinding or milling, are also used. However, such methods have twopossible disadvantages. Firstly, hot spots can develop in the materialbeing ground or the temperature of the material increases. If proteinsor enzymes are the desired product from the biological material, suchheat (whether general or localised) can seriously degrade or render thedesired products inactive. Secondly, on some cells, particularlybotanical cells with very strong cell walls, this method does not alwaysrupture the cell wall.

Methods using the application of a magnetic or electromagnetic field tothe cells are also capable of disrupting cell walls. However this methodis not always commercially reliable or versatile.

A further, known method of cellular disruption is that as discussed inWO 97/05787 (Ashourian). This publication discloses the use of anhomogeniser to homogenize fruit cells within a puree or fluid or toproduce same to reduce the size of cell components under pressure.However, the disruption is disclosed as producing a puree or anhomogenised fluid. The process appears to be inapplicable tointra-cellular disruption of particulate material.

In all the above methods of cell wall disruption the result is a rip ortear in the cell wall. This is not always a break or disruption ofsufficient size to release the contents of the cell for easy access tothe cytoplasm and intact nucleus and organelles. Further, the abovemethods do not result in an homogenous mixture.

An object of the present invention is the provision of apparatus and amethod for the production of a mixture containing disrupted biologicalmaterial. A further object of the invention is the provision of suchapparatus and method which overcome the disadvantages of known methodsof disruption of biological material as described above, with referenceto cells with rigid cell walls or non-rigid and non-elastic cell walls.

DISCLOSURE OF INVENTION

For the purposes of this specification “biological material” includesbut is not limited to: cells with cell membranes, cells with rigid cellwalls, cells with non-elastic, non-rigid cell walls, non-cellularbiological material, intra-cellular material, unbounded homogenousmaterial, and a combination thereof; all material being biologicalmaterial that is, or can be, rendered particulate in appearance.

The present invention provides a method of disrupting biologicalmaterial, said method including the steps of:

drying particulate biological material;

mixing said particulate material with a gas at a pressure between 4 and800 bar and allowing said mixing to continue until gas penetration ofsome or all of the particulate material is effected;

releasing the pressure explosively and reducing the pressure of theparticulate material to atmospheric at a temperature of not more than400° C.;

collecting the resultant product.

The present invention further provides a method of disrupting biologicalmaterial, said method including:

drying particulate biological material;

mixing said particulate material with a gas at a pressure between 4 and800 bar and allowing said mixing to continue until gas penetration ofsome or all of the particulate material is effected, wherein saidparticulate material is mixed in small portions;

separating a small portion of mixed material and gas and releasing thepressure explosively and reducing the pressure within the small portionof the particulate material to atmospheric at a temperature of not morethan 400° C.;

collecting the resultant product; and

repeating the above three steps, said repetition being in the nature ofa continuous process.

Preferably the above methods produce a homogenous mixture of pieces andcytoplasm. Preferably the methods further include, after the step ofreleasing the pressure explosively, the step of allowing the explosivelydecompressed material to impact on or along a shear cone or wall.

Preferably the pressure is in the range 4 and 30 bar pressure.

Preferably the gas is selected from the group consisting of: air, carbondioxide, nitrogen, helium, hydrogen, argon, neon, helium; and acombination of these. The selection of the gas used is dependent on thematerial to be processed, as the gas needs to be substantially inertwith reference to that material. The selection is also dependent oncommercial availability and cost of the gas(es). Preferably the timeperiod for release of the pressure is less than one second, and morepreferably 0.1 second.

Preferably, the gas penetration of the particulate material has reachedan equilibrium before the pressure is explosively released. In theinstance of particulate material with cell walls, this equilibriumoccurs when the gas is in equilibrium within the cell wall. This time isgenerally between 1 to 10 minutes. However, more time may be used forthis step of the method. Optionally the time for this step is between 1and 3 minutes.

Preferably the particulate material is initially of a size between 0.1to 2000 μm, more preferably 0.1 to 50 μm, and most preferably 0.1 to 20μm. With this range of particle sizes it can be seen that particles thesize of bacteria, viruses, procaryotic, eucaryotic cells and cellularinclusions can be the biological material.

The biological material can be selected from the group including:material with rigid cell walls, cells with non-elastic, non-rigid cellwalls, cells with cellular membranes, non-cellular biological material.Examples of such material include pollens, spirulina and other rigidcell walled unicellular species. The biological material can bebiological material with non-rigid cell walls at room temperature whichwalls become rigid walls or non-elastic, non-rigid walls under extremelylow temperatures. Examples of intra-cellular material or non-cellularmaterial include organelles and nuclei, and shark cartilage. An exampleof such material with cell membranes is green lipped mussel powder.

The temperature range in which the above methods can be performed is−200° to 400° C., more preferably −196° to 40° C., and most preferably−15° to 30° C.

Optionally said methods produce a resultant product with a reduced countof biological contaminants, as compared with that of the startingparticulate material, when the starting material is non-fungal material.The method optionally further includes the step of treating theresultant product with ultra-violet light.

Optionally, when the starting material is fungal, said methods produce aresultant product with an increased cell-forming count.

The present invention further provides apparatus for the disruption ofbiological material (as hereinbefore defined), said apparatus including:

a chamber with a first inlet means for particulate material and a secondinlet means for gases and an outlet means for gases and material, saidchamber being capable of withstanding pressures up to 800 bar; and

collection means attached to said outlet means;

wherein said outlet means includes a valve which is capable of releasingthe pressure within the chamber in one second or less.

The present invention further provides apparatus for the disruption ofbiological material (as hereinbefore defined); said apparatus including:

a chamber with a first inlet means for particulate material and a secondinlet means for gases and an outlet means for gases and material, saidchamber being capable of withstanding pressures up to 800 bar;

collection means attached to said outlet means; wherein

said inlet means for particulate material includes two valves (an innerand an outer valve), each independently operated by an actuator, saidvalves being separated by an inlet chamber which is capable ofwithstanding pressures of up to 800 bar;

said outlet means for gases and material includes two valves (an innerand an outer) each independently operated by an actuator, said valvesbeing separated by an outlet chamber which is capable of withstandingpressures of up to 800 bar;

wherein the outlet valve of the outlet means is capable of releasing thepressure within the outlet chamber in one second or less.

The present invention further provides apparatus for the disruption ofbiological material (as hereinbefore defined), said apparatus including:

a chamber with a first inlet means for particulate material and a secondinlet means for gases and an outlet means for gases and material, saidchamber being capable of withstanding pressures up to 800 bar;

collection means attached to said outlet means; wherein

said inlet means for particulate material includes one valve operated byan actuator;

said outlet means for gases and material includes two valves (an innerand an outer) each independently operated by an actuator, said valvesbeing separated by an outlet chamber which is capable of withstandingpressures of up to 800 bar;

wherein the outlet valve of the outlet means is capable of releasing thepressure within the outlet chamber in one second or less.

Preferably said apparatus operates to produce a homogenous mixture ofcell wall pieces and cytoplasm.

Preferably said apparatus also includes means to vibrate said chamber tofacilitate the mixing of the particulate material and the gas. Thecollection means may be any known means of collecting fine particles,for example: a cyclone dust collector, a dust bag, an electrostatic dustprecipitator and a combination of these. Preferably the collection meanscollects the exploded material under inert or substantially inertconditions.

Preferably the collection means further includes a cone placed withinthe collection means adjacent the outlet from the outlet valve of theoutlet chamber; the position of the cone being such that the particulatematerial is still travelling at considerable speed, as a result of theexplosive decompression, when the material impacts the cone and slidesinto the collection means.

Preferably, any of the embodiments of the apparatus operate in thepressure range 4 to 30 bar pressure.

Optionally, any of the embodiments of the apparatus can be operatedunder axenic conditions. Optionally the above-described apparatus canoperated at temperatures in the range −200° to 400° C. more preferably−196° to 40° C. and most preferably −15° to 30° C.

BRIEF DESCRIPTION OF THE DRAWINGS

By way of example only, preferred embodiments of the present inventionare described in detail with reference to the examples and to theaccompanying drawings, in which:

FIG. 1 is a diagrammatic representation of a first preferred embodimentof the apparatus of the present invention for batch operation of themethod of the present invention;

FIG. 2 is a diagrammatic representation of apparatus of a secondpreferred embodiment of the present invention for continuous operationof the method of the present invention;

FIG. 3 is a diagrammatic representation of apparatus of a thirdpreferred embodiment of the present invention for semi-continuousoperation of the method of the present invention;

FIG. 4 shows a photograph of shark cartilage at a magnification of 100times, taken before (a) and after (b) the operation of the method on thematerial in the second preferred embodiment of the apparatus of thepresent invention; and

FIG. 5 shows a photograph of a pollen grain at a magnification of 500times, taken after the operation of the method on the material in thesecond preferred embodiment of the apparatus of the present invention;

FIG. 6 shows a photograph of spirulina at a magnification of 200 times,taken before (a) and after (b) the operation of the method on thematerial in the second preferred embodiment of the apparatus of thepresent invention; and

FIG. 7 shows a photograph of green lipped mussel powder at amagnification of 10 times, taken before (a) and after (b) the operationof the method on the material in the second preferred embodiment of theapparatus of the present invention.

BEST MODES FOR CARRYING OUT THE INVENTION

Referring to FIG. 1, a chamber 2 is there shown, with a gas inlet pipe3, a powder entry pipe 4 and an outlet pipe 5. The chamber 2 isapproximately cylindrical and capable of withstanding pressures inexcess of 30 bar.

The gas inlet pipe 3 is connected by known means to a gas cylinder (notshown) with known cutoff valving. The gas cylinder may alternatively beany other source of pressurised gas. The gas is any gas which is inertto the powder to be disrupted. Examples of such gases, which arecommercially available at a reasonable cost are: air, nitrogen andcarbon dioxide. However, if so desired, another gas may be used.Examples of such gases include: the noble gases and hydrogen. Acombination of gases may be used, if so desired.

The powder inlet pipe 4 includes a valve 6 capable of withstanding thepressures used in the chamber 2. The outlet pipe 5 includes a rapiddischarge valve 7 of known type (for example, a ball valve) which canwithstand the pressures in the chamber 2 and which can open extremelyrapidly, so that the contents of the chamber 2 can be evacuated in under1 second. In practice it has been found that this time is preferably0.75 second or less.

The outlet pipe 5 is connectable to a collector 8, which can be anyknown type of powder collector into which expanding gases can passquickly. Examples of such collectors include a dust bag, a cyclonecollector, an electrostatic precipitator, and a combination of these.

The chamber 2 is cylindrical is shape and is preferably of a diameterless than 150 millimeters. If so desired, an appropriate, largerpressure vessel could be used. The chamber 2 can rest on a vibrator 9,of known type, if so desired. Such vibrator 9 can be electrically ormechanically driven. The vibrator 9 may be replaced by anothermechanical equivalent, for example springs, if so desired.

The above described apparatus is used as follows: powder enters thechamber 2 through the powder inlet pipe 4, and the inlet valve 6 isclosed. The rapid discharge valve 7 is closed. Gas is introduced intothe chamber 2 through the gas inlet pipe 3. The gas valving is closedwhen the pressure in the chamber 2 is at 30 (or at the pre-determinedpressure) bar. The vibrator 9 operates to keep the gas and the powdermixing in the closed chamber 2.

After a mixing period of between 20 seconds to 10 minutes, the rapiddischarge valve 7 is opened. The powder and gas exit the chamber 2 in anexplosive decompression from the chamber 2. The powder remnants arecollected in the collector 8. The mixing period is sufficient for thegas to penetrate some or all of the particulate material. If, forexample, the particulate material has cell walls, the mixing period issufficient for the gas to penetrate the cell walls and reach anequilibrium pressure there. If so desired, the mixing period may belonger, or shorter.

In practice it has been found that at 10 bar pressure, one kilogram ofmaterial can be efficiently mixed with nitrogen gas in one minute.

The rapid discharge valve 7 preferably permits the explosive removal ofthe gas and powder from the chamber 2 in less than one second. Morepreferably the time is between 0.75 and 0.1 seconds. In the firstpreferred embodiment of the method, the explosive decompression iscarried out at a room temperature or not more than 40° C. If the releaseof organelles, proteins or enzymes from the cells is desired, then theuse of temperatures any greater than this may lead to degradation of thedesired product.

Preferably the collection of the powder remnants in the collector 8 isunder inert or substantially inert conditions. Optionally this isachieved by expelling substantially all the air from the collector 8prior to the opening of the rapid discharge valve 7, and collecting thepowder remnants in the presence of some or all of the expelled gas fromthe chamber 2. Alternatively, the collector 8 may be operated with agaseous atmosphere of pure nitrogen or other inert gas; or operate in astrong vacuum until the rapid discharge valve 7 is opened.

The above described chamber 2 and collector 8 have been described withreference to FIG. 1, which displays the chamber 2 and collector 8 in ahorizontal position. If so desired, the orientation of the chamber 2,collector 8 and associated parts can be vertically oriented.

The powder used includes any of the biological materials as previouslydefined. Examples of such materials include, but are not limited to:plant cells with rigid walls (for example, cells with a skeletalfunction, and spores or pollens); animal cells with fibroid or calcifiedcell membranes or matrices; and bacterial cells.

EXAMPLES

In the examples below the material was dried and ground to a finepowder, if not already in that form, to produce the starting material.The method used was a batch operation at less than 30 bar pressure andat a room temperature between 15° to 30° C. The starting material andthe resultant product were photographed under the same conditions.

Example 1

An example is Spirulina (known as either Spirulina (Arthrospira)platensis or Arthrospira (Spirulina) platensis (“spirulina”)). This iscommonly seen in two species—A (s) platensis and A (s) maxima. FIG. 6ashows powdered spirulina (200 times magnification) which was used as thestarting material. FIG. 6b is the resultant product at the samemagnification.

Example 2

Referring to FIGS. 4a and 4 b, the starting material and the resultantproduct, where the material was shark cartilage is there shown. Thecartilage shown in FIG. 4a was previously milled to a ‘super fine’ levelby a known milling machine.

Example 3

FIG. 5 shows a pollen grain immediately after explosive decompression.The grain is magnified 500 times. The cell wall has been breached inthree places (one of which is under the top edge of the grain).Cytoplasm has been discharged through the breaches.

Example 4

FIG. 7 shows two views (before (a) and after (b)) where the startingmaterial is green lipped mussel powder. The magnification is 10 times.The powder is produced by drying the mussel, and shredding or milling toproduce a fine powder. FIG. 7b shows the resultant product from thepowder of FIG. 7a.

The material used in these examples: spirulina, pollen, shark cartilageand green lipped mussel are all commercially available products.

There are cells, or other biological material, that do not contain arigid cell wall or a cell wall that is non-rigid but also non-elastic.This occurs at normal room or operating temperatures for such cells.Such cell walls, for some material, can be rendered rigid or non-rigidbut also non-elastic, by decreasing the temperature of the material to atemperature in the range −15° to −200°. This can be achieved by loweringthe temperature of the chamber 2 and collector 8 and by reducing thetemperature of the biological material prior to passing it though thechamber 2.

In practice it has been found that with one kg of dried particulatespirulina, the degree of cell wall disruption at varying pressure is asfollows:

4 bar: 40% 7 bar: 65% 10 bar: 90-95%

These results are based on a mixing time of 2 minutes and a release timefor the rapid discharge valve of 0.75 seconds. It has been found inpractice that this method reduces the average particle size from about20μm to 1-2 μm.

In practice it has been found that, when the particulate material iscellular in nature, this method and apparatus disrupts only the cellwalls . The nucleus and other organelles are left intact, but accessibleby standard chemical and biochemical reactions. In practice it has alsobeen found that cell walls are substantially uniformly disrupted, sothat the resultant cell wall particles are in a narrow range of particlesizes. If the nuclei and organelles are dried out these can then bedisrupted by a further passing of the material through the chamber 2.

If so desired, the gas used could also be collected after use andrecycled after appropriate filtering.

The above described method and apparatus have been described withreference to a batch operation. However it will be appreciated that themethod could also be used in a continuous or semi-continuous process(using a reciprocating pump for the pressure variation differentialrequired), without departing from the scope of the invention. Similarly,the same method could be used again on material already passed throughthe batch operation, either in the same equipment, or in a series ofchambers 2.

Referring to FIG. 2, a second preferred embodiment of the apparatus ofthe present invention, for a continuous process operation, isthereshown. Said apparatus includes a chamber 12, a gas inlet pipe 13, apowder entry pipe 14 and an outlet pipe 15. The chamber 12 isapproximately cylindrical and capable of withstanding pressures inexcess of 30 bar.

The gas inlet pipe 13 is connected by known means to a gas cylinder (notshown) with known cutoff valving. The gas cylinder may alternatively beany other source of pressurised gas. The gas is any gas which is inertto the powder to be disrupted, as described above with reference to thefirst preferred embodiment of the invention.

The entry pipe 14 includes an inlet chamber 16 which has a capacity ofless than 5 mL, preferably 3 mL. The inlet chamber 16 is capable ofwithstanding the same pressures as the chamber 12. At each end of theinlet chamber 16 is a valve. The valve connecting the inlet chamber 16to the chamber 12 is the inner inlet valve 21. The valve connecting theinlet chamber 16 to a hopper 22 is the outer inlet valve 20. The valves20, 21 are capable of withstanding the pressures used within the chamber12, 16; for example, ball valves.

The outlet pipe 15 includes an outlet chamber 17 which has a similarvolume and pressure capacity to that of the inlet chamber 16. At eachend of the outlet chamber 17 is a valve. The valve connecting the outletchamber 17 to the chamber 12 is the inner outlet valve 23. The valveconnecting the outlet chamber 17 to a collector 18 is the outlet outervalve 24. The valves 23, 24 are capable of withstanding the pressuresused within the chambers 12, 17; for example ball valves. Additionally,the outer outlet valve 24 is a rapid discharge valve which can be openedextremely rapidly, so that the contents of the outlet chamber 17 can beevacuated to atmospheric pressure in under 1 second. In practice it hasbeen found that this time is preferably 0.75 seconds or less.

The hopper 22 is releasably connected to the upper or outer end of thegas entry pipe 14 and releasably sealed thereto. The hopper 22 is ofknown design for containing particulate material. If so desired, thehopper 22 may include a vibrator or other known means for the preventionof ridging of material within the hopper 22.

The outlet pipe 15 is connectable to the collector 18, which can be anyknown type of powder collector though which expanding gases can passquickly. Examples of such collectors include a dust bag, a cyclonecollector, an electrostatic precipitator, and a combination of these.The collector 18 may further include, positioned within the collector18, a shear wall or shear cone 26. The tip of the shear cone 26 isadjacent the exit of the outer outlet valve 24, with the shear cone 26being aligned to the same axis as the chamber 12.

The valves (20, 21, 23, 24) are each controlled by an actuator 25 ofknown type. Preferably, each actuator 25 is operated pneumatically andcapable of remote computer control. With the positioning of anappropriate level sensor (not shown) in the hopper 22 and on the gasinlet pipe 13, the apparatus can be remotely monitored and controlledelectronically. Each actuator 25 also includes a means to manuallyoverride any automated operation.

The chamber 12 is cylindrical in shape and is preferably of a diameterless than 150 mm. If so desired, an appropriate, larger pressure vesselcould be used. The chamber 12 is shown in FIG. 2 with a vertical axis,but is capable of operation at any angle. The chamber 12 can be vibratedor shaken. If such assistance to mixing within the chamber 12 isrequired, this can be achieved by appropriate operation of four springs19 attached to the external wall of the chamber 12. The second end ofeach of the springs 19 can be attached in known manner to a framework(not shown). The chamber 12 can then be shaken or vibrated in knownmanner. If so desired, other mechanical equivalents to the springs 19may replace the springs 19, as described above with reference to thefirst preferred embodiment.

The above described apparatus works as follows: all valves (20, 21, 23,24) are closed and the chamber 12 brought up to the operating pressureby use of the gas from inlet pipe 13. The attendant valve 13 a is closedso that the chamber 12 is at the normal operating pressure, (for example16 bar). Preferably this is between 10 and 20 bar, depending on thematerial to be ruptured but can be up to 800 bar pressure.

The dry powder to be disrupted is placed in the hopper 22. The outerinlet valve 20 is opened. Any excess gas under pressure is releasedthrough the powder, stirring the powder. Alternatively, if so desired,tubing or piping arrangements (not shown) may be used adjacent the outerinlet valve 20 as a bypass to vent all or most of the expelled gas awayfrom the powder.

A quantity of powder falls (under gravity, or is pushed) into the inletchamber 16. The outer inlet valve 20 is closed and the inner inlet valve21 opened. The powder enters the chamber 12 and the inner inlet valve 21is closed. This operation of the valves 20, 21 continues in a cyclicalprocess, each cycle taking approximately 3 seconds. This time may bevaried, to more or less, as is desired. The variation will depend on therequired average residence time required for the particulate material inthe chamber 12.

The outlet valves (23, 24) also operate a similar cycle. The inneroutlet valve 23 opens, allowing approximately 3 mL of material and gasunder pressure to enter the outlet chamber 17. The inner outlet valve 23is closed and the outer outlet valve 24 is opened, with the material andgas being explosively decompressed into the collector 18. The time forexplosive decompression is less than one second, and preferably between0.1 and 0.75 seconds. Once all gas and material is vented, the outeroutlet valve 24 is closed and the cycle is repeated.

In practice it has been found appropriate for the chamber 12 to have aninternal volume of approximate 750 mL (being 600 mm long and having adiameter of approximately 50 mm). With approximately 3 mL of particulatematerial entering and leaving the chamber 12 every three seconds, theapproximate time of residence of material within the chamber 12 is twoto three minutes. However with appropriate variation of the cycles ofthe inlet and outlet valves (20, 21, 23, 24) this residence time can bevaried between 20 seconds to 10 or more minutes.

For example, the minimum average residence time for a particulatematerial with a cell wall or membrane required is the time taken toachieve an equilibrium pressure of the gas in the cell wall or membrane.The chamber 12 is capable of operating at approximately roomtemperature.

Any variations in the internal pressure of the chamber 12 can bemonitored by known means and the gas inlet valve 13 a operated to allowmore gas to enter the chamber 12, to maintain as close to a constantpressure within the chamber 12, as possible.

The powder remnants, after leaving the outer outlet valve 24, impact onthe shear cone 26. This action aids in tearing apart or shattering theruptured pieces of the material. This assists in obtaining uniformity ofthe piece size in the disruption of the cell walls and leads to a small,consistent range of ruptured particle sizes within the rupturedmaterial.

The manner of collection of the powder remnants is as previouslydescribed. Also, as previously described, the chamber 12 can be vibrated(using the springs 19) to assist the attaining of a gas pressureequilibrium within the cell walls of the particulate material in thechamber 12.

The above described apparatus can be seen to operate a continuous cycle.With the size of chamber 12, as given above, and the described dutycycle of the two sets of valves (20, 21, 23, 24), between 5 to 10kilograms of particulate material can be processed per hour. With aresidence time within the chamber 12 of less than two minutes or alarger chamber (etc), the quantity of material processed may be variedto suit particular requirements.

As with the first preferred embodiment the gas can be recycled, if sodesired.

Whist the above described apparatus is used for a continuous operation,it will be appreciated that batch operations are also possible, withoutdeparting from the scope of the invention.

Referring to FIG. 3 a third preferred embodiment of the apparatus of thepresent invention is thereshown. Unless otherwise specified, likenumbers refer to like parts of the second preferred embodiment of theapparatus. The major difference between the second and third embodimentsis that the inlet chamber (16) and the outer inlet valve 20 are absent.This results in a different, semi-continuous cycle.

The apparatus of the third preferred embodiment operates as follows: thevalves 13 a, 21, 23 are closed so that the chamber 12 is isolated. Theparticulate material is placed in the hopper 22. The inlet valve 21 isopened and the material allowed to enter the chamber 12. Whenapproximately 600 mL of material have entered the chamber the inletvalve 21 is closed.

The gas inlet valve 13 a is opened and the chamber 12 is pressurised tothe pre-determined required pressure (as described previously above).The material is left in the chamber 12 for a minimum of between 1 to 10minutes, preferably 2 minutes. If so desired, the material may remain inthe chamber 12 for longer than is the minimum required.

The outlet chamber 17 and outlet valves 23, 24 them operate in the samemanner as is described for the second preferred embodiment. The onedifference is that the outlet chamber 17 is larger in size, being in therange 3 to 10 mL in size.

The cycle is repeated until all the material in the chamber 12 isemptied through the outlet chamber 17. The entire process described isthen repeated. As is desired, new material may be added to the hopper 22for each cycle, or material may remain within the hopper 22 during eachcycle.

Within this semi-continuous process, given the dimensions above for thechamber and the residency time, each cycle takes approximately 6 to 10minutes. Thus, with a material having a specific gravity of 0.5 it ispossible to process up to 3 kg per hour of material.

Optionally, and if so desired, any of the preferred embodimentsdescribed or referred to herein incorporate the encasing of the chamber(2, 12) in an impact-resistant plastic (not shown) of known type.

The operation of the first preferred embodiment at lowered temperaturescan also be achieved for the second and third embodiments by loweringthe temperature of the chamber 12 and collector 18 and by reducing thetemperature of the biological material prior to passing it though thechamber 12. The temperature ranges of the first preferred embodiment canbe applied in the second and third embodiments.

The apparatus of any embodiment is capable of being operated underaxenic conditions. It has been found that, with the selection ofappropriate starting biological material, the resultant product may havea reduced contamination count as compared with the count for the driedparticulate starting material. However the biological material needs tobe non-fungal material.

With all the preferred embodiments of the present invention as describedor incorporated herein, it will be appreciated that the apparatus mayinclude means to ground or earth all the component parts of theapparatus for the avoidance of static build-up and to remove thepotential for a dust explosion.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

What is claimed is:
 1. An apparatus for disrupting dried particulatebiological material, said apparatus comprising: a chamber with a firstinlet means for said material and a second inlet means for gases and anoutlet means for gases and material, said chamber being capable ofwithstanding pressures up to 800 bar; collection means attached to saidoutlet means; said outlet means for gases and material includes an innervalve and an outer valve, each said valve independently operated by anactuator, said valves being separated by an outlet chamber which iscapable of withstanding pressures of up to 800 bar; and said outer valveis capable of releasing the pressure within the outlet chamber in onesecond or less.
 2. An apparatus for disrupting dried particulatebiological material, said apparatus comprising: a chamber with a firstinlet means for said material and a second inlet means for gases and anoutlet means for gases and material, said chamber being capable ofwithstanding pressures up to 800 bar; and collection means attached tosaid outlet means; said outlet means includes at least one outlet valve,which is capable of releasing the pressure within the chamber in onesecond or less; and said first inlet means includes an inner inlet valveand an outer inlet valve, each independently operated by an actuator,said inlet valves being separated by an inlet chamber which is capableof withstanding pressures of up to 800 bar.
 3. The apparatus as claimedin claim 2 wherein said outlet means for gases and material includes aninner outlet valve and an other outlet valve each independently operatedby an actuator, said outlet valves being separated by an outlet chamberwhich is capable of withstanding pressures of up to 800 bar.
 4. Theapparatus as claimed in claim 1 wherein said apparatus further includesa shear cone or wall immediately adjacent said outlet means.
 5. Theapparatus as claimed in claim 2 wherein said apparatus further includesa shear cone or wall immediately adjacent said outlet means.
 6. Theapparatus as claimed in claim 1 wherein said material is selected fromthe group consisting of: cells with cell membranes, cells with rigidcell walls, cells with non-elastic cell walls, cells with non-rigid cellwalls, non-cellular biological material, intra-cellular material,unbounded homogenous material, shredded biological material and acombination thereof.
 7. The apparatus as claimed in claim 2 wherein saidmaterial is selected from the group consisting of: cells with cellmembranes, cells with rigid cell walls, cells with non-elastic cellwalls, cells with non-rigid cell walls, non-cellular biologicalmaterial, intra-cellular material, unbounded homogenous material,shredded biological material and a combination thereof.
 8. The apparatusas claimed in claim 7 wherein said cells with rigid cell walls includepollens and spirulina.
 9. The apparatus as claimed in claim 7 whereinsaid cells with rigid cell walls include pollens and spirulina.
 10. Theapparatus as claimed in claim 1 wherein said apparatus is operated underconditions selected from the group consisting of: axenic conditions,inert conditions, and a combination thereof.
 11. The apparatus asclaimed in claim 2 wherein said apparatus is operated under conditionsselected from the group consisting of: axenic conditions, inertconditions, and a combination thereof.
 12. The apparatus as claimed inclaim 1 wherein the collection means is selected from the groupconsisting of; a filter, a cyclone dust collector, a dust bag, anelectrostatic dust precipitator and a combination thereof.
 13. Apparatusas claimed in claim 2 wherein the collection means is selected from thegroup consisting of: a filter, a cyclone dust collector, a dust bag, anelectrostatic dust precipitator and a combination thereof.